Pyrrolidine compounds and methods for selective inhibition of dipeptidyl peptidase-IV

ABSTRACT

The present invention is directed to pyrrolidinylaminoacetyl pyrrolidine boronic acid compounds that display selective, potent dipeptidyl peptidase IV inhibitory activity. These compounds are useful for the treatment of disorders that can be regulated or normalized via inhibition of DPP-IV including those characterized by impaired glycemic control such as Diabetes Mellitus and related conditions. The compounds can be administered alone or with another medicament that displays pharmacological activity for treatment of these and other diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 11/381,085, filed May 1, 2006, now U.S. patent application Ser. No.7,317,109, issued on Jan. 8, 2008, which is a continuation-in-part ofU.S. application Ser. No. 10/514,575, filed on Oct. 27, 2005, now U.S.patent application Ser. No. 7,674,913, issued on Mar. 9, 2010, which isa national stage application of PCT/US04/037820, filed Nov. 12, 2004,which claims priority to U.S. provisional application No. 60/519,566,filed on Nov. 12, 2003; U.S. provisional application No. 60/557,011,filed on Mar. 25, 2004; and U.S. provisional application No. 60/592,972,filed on Jul. 30, 2004. This application is also a continuation-in-partof U.S. Application No. 60/676,808, filed on May 2, 2005. Theseapplications are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a pyrrolidinylaminoacetyl pyrrolidineboronic acid compound and its use as a selective inhibitor ofpost-proline/alanine cleaving amino-dipeptidases, particularlydipeptidyl peptidase-IV (DPP-IV). The invention also relates tomethodology for employing a pyrrolidine compound, alone or with anothermedicament, to treat a DPP-IV-related disease, including but not limitedto disorders characterized by impaired glycemic control, especiallyDiabetes Mellitus and related conditions. Thus, the invention hasapplications in the medicinal, chemical, pharmacological, and medicalfields.

BACKGROUND OF THE INVENTION

Dipeptidyl peptidase-IV (DPP-IV) is a serine protease that belongs to agroup of post-proline/alanine cleaving amino-dipeptidases. DPP-IVcatalyzes the release of an N-terminal dipeptide of any configurationfrom proteins, and preferably, the dipeptide contains an N-terminalpenultimate proline or alanine.

The physiological role of DPP-IV has not been established fully. It isbelieved to play an important role in regulatory peptide metabolism,which, among other things, controls various physiological functionsincluding but not limited to glycemic control and insulin sensitivity.In particular, DPP-IV has been implicated in the control of glucosemetabolism because its substrates include the insulinotropic hormones,glucagon like peptide-1 (GLP-1) and gastric inhibitory peptide (GIP),which are inactivated by removal of their two N-terminal amino acids.

In vivo administration of synthetic inhibitors of DPP-IV preventsN-terminal degradation of insulinotropic hormones including, GLP-1 andGIP, resulting in higher plasma concentrations of these hormones,increased insulin secretion and, consequent improved glucose tolerance.Therefore, such inhibitors have been proposed for the treatment ofpatients with impaired glycemic control such as Diabetes Mellitus andrelated conditions

This proposal has significant difficulties, however. Additionaldipeptide cleaving amino-dipeptidases have also been discovered,including DPP-VII, DPP-VIII, DPP-IX, and fibroblast activation protein(FAP), which can have substrate and inhibitor specificity similar toDPP-IV. The precise physiological role of each of these dipeptidecleaving enzymes is not well defined. But, their propensity to cleaveN-terminus dipeptides from proteins in general indicates that theseamino-dipeptidases are involved in many physiological cycles. Thus, thedifficulty concerning inhibitors of DPP-IV is that they can also affectthe other members of the enzyme group. The evidence indicates that, forexample, other inhibitors of DPP-IV, which also inhibit the otheramino-dipeptidases such as DPP-VIII, will cause toxic effects inanimals.

Accordingly, a need exists for compounds that are useful for inhibitingDPP-IV without an adverse event profile that precludes chronicadministration.

SUMMARY OF THE INVENTION

The present invention is directed to a selective DPP-IV inhibitor andmethods of use that are effective in treating conditions that may beregulated or normalized by inhibition of DPP-IV. More particularly, theinvention is directed to a pyrrolidinylaminoacetyl pyrrolidine boronicacid compound. This pyrrolidinylaminoacetyl pyrrolidine boronic acidcompound is useful at effective doses for treatment of malconditionsassociated with DPP-IV activity and is a selective inhibitor of DPP-IV.

A pyrrolidinylaminoacetyl boronic acid compound of the invention(hereinafter the pyrrolidine compound of the invention) has a structurerepresented in part by Formula I.

The substituents and bond designations of formula I include R² and R³,which, independently or together, are —OH, —O⁻M⁺ wherein M⁺ is a cation,a hydroxyl bearing a boronic acid protecting group, or a group capableof being hydrolyzed to a hydroxyl group in an aqueous solution atphysiological pH or in biological fluids; and the wavy lines atasymmetric carbons C^(a) and C^(b), which independently indicate foreach asymmetric carbon an R configuration, an S configuration, or amixture of both configurations such that all stereoisomers and allstereomeric mixtures are included. Also included within the scope of theinvention are a cyclic isomer thereof, any pharmaceutically acceptablesalt thereof, any prodrug thereof, and any solvate thereof.

A pyrrolidine compound of the invention may exist in either of twoforms, the linear form represented by formula I above and the cyclicisomer form represented by formula V below.

The cyclic isomer form and the linear form are in thermodynamicequilibrium when in solution. The equilibrium shifts depending upon pH.Thus, the predominance of one form over the other in solution dependsupon the pH so that at acidic pH, the linear isomer predominates whileat basic pH, the cyclic isomer predominates. The linear and cyclicisomers are also stable such that either form may be isolated as asolid. The isolated cyclic isomer can function as a prodrug.

The invention also is directed to a pharmaceutical compositioncontaining a pyrrolidine compound of the invention and a pharmaceuticalcarrier. The pharmaceutical composition may be formulated to be dosed byany administrative route including but not limited to parenteralinjection, oral, buccal, rectal and the like.

The invention is as well directed to a method of treatment of amalcondition that can be regulated or normalized via inhibition ofDPP-IV. The method involves administration of an effective amount of apyrrolidine compound of the invention, such as would be present in apharmaceutical composition of the invention, to mammals, especiallyhumans, to affect a malcondition that can be regulated or normalized viainhibition of DPP-IV. Preferably, an effective amount of a pyrrolidinecompound of the invention exhibits lower toxicity than do non-selectiveinhibitors of DPP-IV, particularly in comparison to boronic acidinhibitors of DPP-IV that also display inhibition of other DPP enzymesand FAP. Therefore, the invention is directed to methods for selectivelyinhibiting DPP-IV including administering to a patient in need of suchtreatment a therapeutically effective amount of a pyrrolidine compoundof the invention.

The invention further is directed to a pharmaceutical combination of apyrrolidine compound of the invention and one or more other medicamentsthat are useful for treatment of a malcondition that can be regulated ornormalized via inhibition of DPP-IV. Such malconditions are associatedwith impairments in glycemic control especially Diabetes Mellitus andrelated conditions. A pharmaceutical combination may be formulatedaccording to the invention as a pharmaceutical composition.

The invention is also directed to a process for preparing a pyrrolidinecompound of the invention, a method for preparing a pharmaceuticalcomposition of the invention, and the use of a pyrrolidine compound ofthe invention in a method for the preparation of a medicament fortreating a malcondition that can be regulated or normalized viainhibition of DPP-IV.

DEFINITIONS

The term “absolute configuration” in connection with an asymmetriccarbon is determined by considering the tetrahedral shape of theasymmetric carbon bonds, assigning a priority of 1 through 4 to each ofthe groups bound to the asymmetric carbon with the group having thehighest atomic number having the first priority. If the tetrahedron isviewed from a side remote from group 4, an R absolute configuration isassigned when groups 1-3 are in a clockwise arrangement and an Sabsolute configuration is assigned when groups 1-3 are in acounterclockwise arrangement.

The term “asymmetric carbon” means a carbon atom covalently bound tofour different groups.

The term “beta cell degeneration” is intended to mean loss of beta cellfunction, beta cell dysfunction, and death of beta cells, such asnecrosis or apoptosis of beta cells.

The term “Diabetes Mellitus and related conditions” refers to Type 1diabetes, Type 2 diabetes, gestational diabetes, MODY, impaired glucosetolerance, impaired fasting glucose, hyperglycemia, impaired glucosemetabolism, insulin resistance, obesity, diabetic complications, and thelike.

The term “diabetic complications” refers to conditions, diseases andmaladies associated with diabetes including retinopathies, neuropathies,nephropathies, cardiomyopathies, dermopathies, arthrosclerosis, coronaryartery disease and other known complications of diabetes.

The term “diastereomer” means one member of a group of two or morestereoisomers having at least two asymmetric carbons such that thesestereoisomers are not mirror images of each other.

The terms “DPP-VII, DPP-VIII, DPP-IX and FAP” mean respectively aminodipeptidyl peptidase VII, VIII, IX and fibroblast activation protein.The DPP enzymes cleave dipeptide moieties at the N-terminus of theirprotein or oligopeptide substrates. In particular, the term “DPP-IV”denotes dipeptidyl peptidase IV (EC 3.4.14.5; DPP-IV), also known as“CD-26.” DPP-IV preferentially cleaves a dipeptide from the N terminusof a polypeptide chain containing a proline or alanine residue in thepenultimate position.

The term “enantiomer” means one member of a pair of stereoisomers havingthe same molecular structure and at least one asymmetric carbon suchthat the stereoisomers of the pair are the mirror images of each other.If the enantiomer contains two or more asymmetric carbons, theenantiomeric pair will have opposing asymmetry at each asymmetriccarbon.

The term “group that can be hydrolyzed to a hydroxyl” as used hereinrefers to an ester group formed from the combination of an aliphatic oraromatic alcohol or diol and a boronic acid.

The term “inhibitor” (and its corresponding verb and gerund) means acompound that will reversibly, irreversibly or temporarily interact withan enzyme so as to reduce, modify, slow down or block its enzymaticactivity upon its normal substrate. The interaction may occur within orat the enzymatic site or at an allosteric site associated with theenzyme.

The term “N-protecting group” or “N-protected” as used herein refers tothose groups intended to protect the N-terminus of an amino acid orpeptide or to protect an amino group against undesirable reactionsduring synthetic procedures. Commonly used N-protecting groups aredisclosed in T. W. Greene, P. G. Wuts, “Protective Groups In OrganicSynthesis, 3^(rd) Ed.” (John Wiley & Sons, New York (1999)), which ishereby incorporated by reference. N-protecting groups include acylgroups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl,2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl,4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and the like; sulfonylgroups such as benzenesulfonyl, p-toluenesulfonyl and the like;carbamate forming groups such as benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl,t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl,fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and thelike; alkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl andthe like; and silyl groups such as trimethylsilyl and the like.Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl,t-butylacetyl, phenylsulfonyl, benzyl, 9-fluorenylmethyloxycarbonyl(Fmoc), t-butyloxycarbonyl (Boc) and benzyloxycarbonyl (Cbz).

The term “optically active” means an organic compound containing atleast one asymmetric carbon such that a solution of the organic compoundwill rotate plane polarized light.

The term “optically active mixture” means a mixture of optically activecompounds in solution that will rotate plane polarized light. Theoptically active mixture may be a mixture of diastereomers or an unequalmixture of enantiomers.

The term “pharmaceutical salt” means a salt with an inorganic base,organic base (including basic amino acids), inorganic acid, and organicacid (including acidic amino acids). Included as examples of inorganicbases are alkali metals such as lithium, sodium or potassium; alkalineearth metals such as calcium and magnesium or aluminum; and ammonia.Included as examples of organic bases are trimethylamine, triethylamine,pyridine, picoline, ethanolamine, diethanolamine, and triethanolamine.Included as examples of inorganic acids are the instant inventionincludes, for example, hydro-halogen acids such as hydrochloric acid,hydroboric acid, nitric acid, sulfuric acid, and phosphoric acid.Included as examples of organic acids are mono, di and tri carboxylic orsulfonic acids of 1 to 20 carbons, optionally containing 1 to 6 hydroxylgroups. Included as examples of basic amino acids are arginine, lysineand ornithine. Included as examples of acidic amino acids are asparticacid and glutamic acid. Further examples of pharmaceutically acceptablesalts include the pharmaceutically acceptable salts listed in Journal ofPharmaceutical Science, 66, 2 (1977) which are known to the skilledartisan.

The term “prodrug” means a pharmaceutically acceptable compound thatwill convert to the active ingredient or an active metabolite thereofupon administration of the prodrug to a living organism, preferably amammal, more preferably a human. The conversion may occur by enzymaticaction, chemical hydrolysis, oxidation, reduction or any other in vivophysiological process for chemical or biochemical reaction.

The term “racemic mixture” means an enantiomeric pair of equalproportions such that they cancel each other's rotation of planepolarized light.

A singular term such as “a pyrrolidine compound of the invention”includes the plural such as the various species of the pyrrolidinecompound of the invention as well as mixtures thereof. A plural termsuch as “pyrrolidine compounds of the invention” includes the individualspecies as well as the plural indicated by this term, and also mixturesthereof.

The term “selectivity ratio” refers to the IC₅₀ value generated in abiochemical assay measuring inhibition of DPP-IV compared to the IC50value generated in a biochemical assay measuring inhibition of anotherDPP family member (e.g. DPP-VII, DPP-VIII, DPP-IX or FAP) whereby theratio is obtained by dividing the IC₅₀ value of DPP-VII, DPP-VIII,DPP-IX or FAP by the IC₅₀ value for DPP-IV.

The term “solvate” means a solid, crystalline form of a compound whichalso incorporates molecules of a solvent into the crystal structure.Organic solvents as well as water are included. Another description of awater solvate is a hydrate or hydrated form.

The term “stereoisomer” means one of the absolute configurations of asingle organic molecule having at least one asymmetric carbon. Includedwithin the definition of a stereoisomer are enantiomers anddiastereomers. One stereoisomer has one absolute configuration abouteach of the asymmetric carbons of the organic molecule. An organicmolecule with one asymmetric carbon presents two stereoisomers. Anorganic molecule with two asymmetric carbons presents fourstereoisomers. An organic molecule with three asymmetric carbonspresents eight stereoisomers. Projecting plane polarized light through asolution containing one stereoisomer will cause rotation of thepolarized plane.

The term “stereomeric mixture” means a mixture of two or morestereoisomers and includes enantiomers, diastereomers and combinationsthereof. The stereomeric mixture may or may not be optically active.

The term “stereomeric purity” at a given percentage means that thedesignated stereoisomer predominates at that given percentage in amixture of stereoisomers.

Unless otherwise specifically stated, the definitions of terms forchemical groups, functional groups, moieties and chemical reactionsdescribed herein follow the definitions provided in such organicchemistry textbooks and treatises as “Basic Principles of OrganicChemistry”, Roberts and Caserio, W.A. Benjamin & Co. New York, N.Y.,1965; “Advanced Organic Chemistry”, 4^(th) edition, Jerry March, WileyInterscience, New York, N.Y. 1992; T. W. Greene, P. G. Wuts, “ProtectiveGroups In Organic Synthesis, 3^(rd) Ed.” (John Wiley & Sons, New York(1999), and Hawley's Condensed Chemical Dictionary, 11^(th) Ed., Sax andLewis, Van Nostrand, Reinhold, New York, N.Y., 1987. Moreover, thedefinitions for stereochemical terms are based upon “Stereochemistry ofCarbon Compounds”, Ernest Eliel, McGraw-Hill publisher, New York, N.Y.1962. The disclosures of these text books are incorporated herein byreference.

DETAILED DESCRIPTION OF THE INVENTION

A pyrrolidinylaminoacetyl pyrrolidine boronic acid compound of theinvention (pyrrolidine compound of the invention) has the linear orcyclic isomer structure depicted by formulas I and V above. Allstereoisomers, stereomeric mixtures, pharmaceutically acceptable salts,solvates and prodrugs are included as embodiments of the “pyrrolidinecompounds of the invention” (which is also termed “pyrrolidine compound”or “pyrrolidine compounds” herein). A pharmaceutical compositionincluding a pyrrolidine compound of the invention and a pharmaceuticallyacceptable pharmaceutical carrier is an additional aspect of theinvention. Also included is a pharmaceutical combination of apyrrolidine compound of the invention and a second medicament known tobe useful in the treatment of malconditions characterized by impairedglycemic control, especially Diabetes Mellitus and related conditions. Apharmaceutical combination may be formulated as a pharmaceuticalcomposition of the pyrrolidine compound, the second medicament and apharmaceutically acceptable carrier.

The embodiments of the pyrrolidine compound of the invention include theborate esters and boronic acid groups such that R² and R³ of formulas Iand V, independently or together, are —OH, —O⁻M⁺ wherein M⁺ is a cation,a hydroxyl bearing a boronic acid protecting group, or a group capableof being hydrolyzed to a hydroxyl group in an aqueous solution atphysiological pH or in biological fluids. When R² and R³ are groups thatcan be hydrolyzed to hydroxyls, they may be formed from mono-alcoholsand diols of 1 to 15 carbons and the alcohols and diols may be linear,branched, cyclic or aromatic, and may optionally be substituted withester and/or amide groups. Examples include (+)-pinanediol; pinacol;1,2-dicyclohexyl-ethanediol; 1,2-ethanediol; 2,2-diethanolamine;1,3-propanediol; 2,3-butanediol, diisopropyl tartrate; 1,4-butanediol;diisopropylethanediol; (S,S,)-5,6-decanediol;1,1,2-triphenyl-1,2-ethanediol;(2R,3R)-1,4-dimethyoxy-1,1,4,4-tetraphenyl-2,3-butanediol; methanol;ethanol; isopropanol; catechol; or 1-butanol.

Surprisingly, it has been discovered that a pyrrolidine compound of theinvention displays selectivity for DPP-IV relative to other dipeptidylpeptidase enzymes. By selectivity for DPP-IV it is meant that thepyrrolidine compound of the invention more strongly inhibits DPP-IV,than at least one closely related enzyme such as DPP-VII, DPP-VIII,DPP-IX and FAP. While not wishing to be bound by any theory, it isbelieved that this unexpected selectivity for DPP-IV results in animproved therapeutic profile with diminished side-effects for thepyrrolidine compound of the invention compared with other boronic acidinhibitors and compared to any other non-selective DPP-IV inhibitor. Inparticular it is believed that potent inhibition of DPP-VIII by otherinhibitors correlates with the acute toxicity observed in animalstudies. As detailed in the following Examples section, administering acompound with DPP-IV and DPP-VIII inhibitory activity to dogs leads tosevere emesis and diarrhea. The same compounds administered to somestrains of rats resulted in death. The pyrrolidine compound of theinvention avoids significant inhibition of DPP-VIII and therefore avoidsthe toxicities observed in both dog and rat.

Synthesis of the Pyrrolidine Compounds of the Invention

The invention also relates to processes for preparing a pyrrolidinecompounds of the invention. As shown below and as described in theexamples, the pyrrolidine compound of the invention is prepared byreacting a pyrrolidine, suitably protected with a standard protectinggroup such as Boc-, Fmoc-, Cbz- or the like, with sec-BuLi/TMEDAfollowed by a boron source such as B(OCH₃)₃, to provide the boronicester derivative. Acid hydrolysis of the methyl esters with HCl providesthe boronic acid intermediate 1. Reaction of 1 with (+) pinanediol,deprotection of the amino protecting group, and recrystallizationprovides the pinanediol ester 2 as an isomerically pure salt.

Intermediate 2 is useful for the synthesis of a pyrrolidine compound ofthe invention. For example, N-acylation of 2 with chloroacetyl chlorideprovides the α-chloro amide 3. Treatment of 3 with Na₂CO₃ and apyrrolidinylamine, hydrolysis of the pinanediol boronic ester, andN-deprotection provides a pyrrolidine compound of the invention, 4.

wherein R¹ is hydrogen. The S stereoisomer of 2 or a mixture ofstereoisomers can be obtained by appropriate manipulation of therecrystallization step, which includes a resolution using an opticallyactive pinanediol. Use of an R or S stereoisomer of the aminopyrrolidine reagent for reaction with 3, or use of a stereomeric mixturethereof will yield the desired stereochemistry at the C^(b) asymmetriccarbon as shown in structure 4.

Thus, another aspect of the invention provides a process for preparing apyrrolidine compound of the invention including coupling a reactivecompound of Formula II:

wherein L is a leaving group such as a halogen, mesylate, tosylate,triflate or the like; and R² and R are independently or together are—OH, —O⁻M⁺ wherein M⁺ is a cation, a hydroxyl bearing a boronic acidprotecting group, or a group capable of being hydrolyzed to a hydroxylgroup in an aqueous solution at physiological pH or in biologicalfluids;with a compound of Formula III

wherein Pr is an N-protecting group such as Boc, Cbz, Fmoc, benzyl orthe like; and R¹ is hydrogen; to provide an ester derivative of thepyrrolidine compound of the invention. The resulting boronic esterderivative of the pyrrolidine compound of the invention may bedeprotected to remove Pr and to recover the pyrrolidine compound of theinvention as an ester, a free acid or as a salt. In some embodiments, Lis halogen, including but not limited to Cl. In others R² and R³ areeach methoxy or together are pinanedioxy, e.g. (+)-pinanedioxy as incompound 3 above. In still others, Pr is Boc.

An alternative synthesis of a pyrrolidine compound of the invention isprovided in U.S. patent application Ser. No. 60/704,380, filed Aug. 1,2005 and entitled “Methods of Preparing Hetercyclic Boronic Acids andDerivatives Thereof.”

A pyrrolidine compound of the invention may be formed as apharmaceutically acceptable salt. The pharmaceutical salt may beobtained as the direct product of compound synthesis. In thealternative, the free base may be dissolved in a suitable solventcontaining the appropriate acid (or vice versa), and the salt isolatedby evaporating the solvent or otherwise separating the salt and solvent.The processes for forming a pharmaceutically acceptable salt from anamine compound such as a pyrrolidine compound of the invention arewell-known in the art. See, for example, “The Practice of MedicinalChemistry, Second Edition”, by Camille G. Wermuth, Academic Press, NewYork, N.Y., 1996.

A pyrrolidine compound of the invention may form solvates with standardlow molecular weight solvents, including water to yield hydrates, usingmethods known to the skilled artisan. These processes for formingsolvates are also well-known in the art. See, for example “The Practiceof Medicinal Chemistry” cited above.

It is to be understood that the invention extends to all of thestereoisomers of a pyrrolidine compound, including enantiomers,diastereomers, as well as the racemates and stereoisomeric mixtures. Themixtures may or may not be optically active.

In some embodiments, a pyrrolidine compound of the invention may have anoptical purity of at least about 55%, preferably 80%, more preferably 90wt %, most preferably 98% or more of a single stereoisomer. In otherembodiments, the pyrrolidine compound is an optically-enrichedenantiomer. In still other embodiments, the pyrrolidine compound is amixture of stereoisomers including but not limited to unequal mixturesof enantiomers and/or mixtures of diastereomers.

Method/Use of a Pyrrolidine Compound of the Invention

The method of treatment and use of a pyrrolidine compound of theinvention is based upon inhibition of dipeptidyl peptidase-IV by contactof the enzyme, dipeptidyl peptidase-IV, with a pyrrolidine compound inany of its forms as described above. The contact may be accomplished invitro such as through a diagnostic test or a screening test, or in vivothrough an appropriate administrative route as discussed below.

The in vivo methods according to the invention involve a pyrrolidinecompound of the invention in its role as a selective inhibitor ofDPP-IV. For example, the invention provides a method of treatment of amammal (such as a human) suffering from a malcondition that can beregulated or normalized via inhibition of DPP-IV such as anymalcondition characterized by impaired glycemic control, especiallyDiabetes Mellitus and related conditions by administering an effectiveamount of a pyrrolidine compound of the invention to treat, control,ameliorate or prevent the malcondition. These malconditions are known tobe the result, at least in part, of the presence, or altered activity,of peptides regulated by the enzyme DPP-IV, especially in the context ofits physiological role in glycemic control. These methods of theinvention are accomplished by administering to the mammal (e.g., ahuman) an effective amount of a pyrrolidine compound of the invention.Treatment is affected by inhibition of DPP-IV. Administration istypically accomplished through use of a pharmaceutical compositioncontaining a pyrrolidine compound of the invention.

The method of the invention further includes a method for selectivelyinhibiting DPP-IV over related enzymes. In some embodiments of themethods for treatment, DPP-IV is inhibited by greater than 5-foldrelative to one or more other dipeptidyl peptidases. In otherembodiments, DPP-IV is inhibited by greater than 10-, 20-, or even50-fold or more over other dipeptidyl peptidases. Exemplary otherdipeptidyl peptidases include DPP-VII, DPP-VIII, DPP-IX, and FAP. Forexample, a pyrrolidine compound of the invention can selectively inhibitDPP-IV over dipeptidyl peptidase-VII, or DPP-IV over dipeptidylpeptidase-VIII, or DPP-IV over dipeptidyl peptidase-IX, or DPP-IV overfibroblast activation protein (FAP). In additional embodiments, apyrrolidine compound of the invention selectively inhibits DPP-IV overdipeptidyl peptidase-VIII and fibroblast activation protein. In otherembodiments, the pyrrolidine compound of the invention selectivelyinhibits DPP-IV over dipeptidyl peptidase-VII, dipeptidylpeptidase-VIII, and fibroblast activation protein. This selectivityapplies to in vitro and to in vivo situations. In particular, it hasbeen determined in an in vivo protocol study in humans that apyrrolidine compound of the invention maintained selectivity forinhibition of DPP-IV over the other amino dipeptidyl peptidases.Preferably, the DPP-IV selectivity is shown relative to DPP-VIII.

For in vivo use as a DPP-IV inhibitor, a pyrrolidine compound of theinvention may be formulated in any manner as described herein andadministered in an effective amount to a patient (human) suffering froma malcondition that can be regulated or normalized by inhibition ofDPP-IV, especially a malcondition characterized by impaired glycemiccontrol, especially Diabetes Mellitus and related conditions. Forexample, the malcondition can be Type 1 diabetes, Type 2 diabetes,gestational diabetes, MODY, impaired glucose tolerance, impaired fastingglucose, hyperglycemia, impaired glucose metabolism, impaired glucosetolerance (IGT) and its progression to Type II diabetes,hyperinsulinemia, obesity, beta cell degeneration (in particularapoptosis of beta cells), the progression of non-insulin-requiring TypeII diabetes to insulin requiring Type II diabetes; loss of the numberand/or the size of beta cells in a mammalian subject, and diabeticcomplications such as retinopathy, neuropathy, nephropathy,cardiomyopathy, dermopathy, diabetes related infection, atherosclerosis,coronary artery disease, stroke and similar malconditions.

In other embodiments of method of treatment according to the invention,insulin resistance is a component of the malcondition that can beregulated or normalized by inhibition of DPP-IV. For example, themalconditions can be impaired fasting glucose, impaired glucosetolerance, polycystic ovarian syndrome and the like. In yet otherembodiments, the malcondition that can be regulated or normalized byinhibition of DPP-IV involves a decrease of islet neogenesis, β-cellsurvival, or insulin biosynthesis.

The administered dose of a pyrrolidine compound of the invention will becarefully adjusted according to age, weight and condition of thepatient, as well as the route of administration, dosage form and regimenand the desired result. The ultimate choice of dosage, route andpharmaceutical formulation will determined by the patient's attendingphysician, whose wisdom and judgment will guide this process. The dosefor adults may range from about 0.5 to about 2,000 mg per day,preferably about 10 mg to about 1000 mg per day, more preferably about50 mg to about 750 mg per day which can be administered in a single doseor in the form of multiple doses given up to 4 times per day.

The use of a pyrrolidine compound of the invention also includes themanufacture of a medicine and a method of treatment using such amedicine in the form of a pharmaceutical composition.

Pharmaceutical Combinations and Their Use in Treatment

A pyrrolidine compound of the invention may be combined with a secondmedicament to form a pharmaceutical combination of the invention. Thesecond medicament is a known agent for treating, controlling, orpreventing a malcondition that can be regulated or normalized viainhibition of DPP-IV. The malconditions treated by such combinations arethose that can be regulated or normalized via inhibition of DPP-IV andthus are the same as those described above in connection with soletreatment for a pyrrolidine compound of the invention.

The second medicament may include a therapeutically effective amount ofa dipeptidyl peptidase-IV inhibitor other than the pyrrolidine compoundof the invention. The second medicament preferably may be a knownanti-diabetic agent including but not limited to an agent that increasesinsulin secretion, an agent that increases insulin sensitivity, an agentthat reduces the uptake of sugar from the gastrointestinal track, anagent that enhances the effect of endogenous peptides or proteins thatplay a role in glycemic control, or an agent that acts a replacementtherapy for endogenous peptides or proteins that have a known role inglycemic control. Such agents include but are not limited to glyburide(e.g. Micronase and Diabeta), glipizide (e.g. Glucotrol), nateglinide(e.g. Starlix), repaglinide (e.g. Prandin), metformin (e.g. Glucophage),rosiglitazone (e.g. Avandia), acarbose (e.g. Precose), miglitol (e.g.Glyset), exenatide (e.g. Byetta), and insulin (e.g. Humulin andNovolin). Additional exemplary agents include but are not limited tobiguanides, chlorpropamide, a glucagon-like peptide-1 (GLP-1) or mimeticthereof such as LY315902 or LY307161, glimepiride, meglitinide,phenformin, pioglitazone, sulfonyl ureas, troglitazone, G1-262570,isaglitazone, JTT-501, NN-2344, L895645, YM-440, R-119702, AJ9677,KAD1129, APR-HO39242, GW-409544, KRP297, AC2993, Exendin-4, and NN2211.The chemical structures, trivial names and pharmacological studies ofthe foregoing compounds designated by letters and numbers are readilyavailable from the web, for example, by entering the letter/numberdesignation as a search term in the GOOGLE search web site.

A pyrrolidine compound of the invention may be used in combination withone or more second medicaments useful as antidiabetic agents (employedto treat diabetes and related diseases). The second medicament may beadministered orally in the same dosage with the pyrrolidine compound ofthe invention, or in a separate oral dosage form. The pyrrolidinecompound of the invention and the second medicament may also beadministered, for example by injection, separately, simultaneously or asa mixture.

The pharmaceutical combination of the invention can be formulated as apharmaceutical composition of a pharmaceutically acceptable carrieralong with a pyrrolidine compound of the invention and one or moresecond medicaments.

In the pharmaceutical combination of the invention, the pyrrolidinecompounds of the invention are typically present in a weight ratio tothe second medicament of from about 0.01:1 to about 100:1, orpreferentially from about 0.1:1 to about 5:1.

The use of a pyrrolidine compound of the invention in combination withone or more other antidiabetic agents may produce antihyperglycemicresults greater than that possible from each of these antidiabeticagents alone. The use of a pyrrolidine compound of the invention incombination with one or more other antidiabetic agents may also producea synergistic effect in that the antihyperglycemic result may be greaterthan the combined additive antihyperglycemic effects produced by theseantidiabetic agents.

The effective amount of a second medicament formulated as a component ofthe pharmaceutical combination of the invention will follow therecommendations of the second medicament manufacturer, the judgment ofthe attending physician and will be guided by the protocols andadministrative factors for amounts and dosing as indicated in thePHYSICIAN'S DESK REFERENCE (PDR).

The administered dose of a pyrrolidine compound of the invention withinthe pharmaceutical combination will be carefully adjusted according toage, weight and condition of the patient, as well as the route ofadministration, dosage form and regimen and the desired result. Theultimate choice of dosage, route and pharmaceutical formulation willdetermined by the patient's attending physician, whose wisdom andjudgment will guide this process.

The compositions described above may be administered in the dosage formsas described above in single or divided doses of one to four timesdaily. It may be advisable to start a patient on a low dose combinationand work up gradually to a high dose combination.

In a further embodiment of the pharmaceutical combinations of theinvention, the second medicament can be known anti-obesity agentsincluding but not limited to a beta 3 adrenergic agonist, a lipaseinhibitor, a serotonin (and dopamine) reuptake inhibitor, a thyroidhormone receptor-beta agonist, an anorectic agent, a fatty acidoxidation upregulator, or a mixture of any two or more thereof.Suitable, known anti-obesity agents include orlistat, sibutramine,topiramate, axokine, dexamphetamine, phentermine, phenylpropanolamine,famoxin, mazindol, or a mixture of any two or more thereof. Theseanti-obesity agents may be employed in the same dosage form with apyrrolidine compound of the invention or in different dosage forms, andthe administrative dosages and regimens applied to the anti-obesityagents will follow the recommendations and guides generally known in theart and/or set out in the PDR.

In another embodiment of the pharmaceutical combinations of theinvention, the second medicament may be an agent for treating polycysticovary syndrome. The agent for polycystic ovary syndrome which may beoptionally employed in combination with a pyrrolidine compound of theinvention may be 1, 2, or more of gonadotropin releasing hormones(GnRH), leuprolide (Lupron®), Clomid®, Parlodel®, oral contraceptives orinsulin sensitizers such as PPAR agonists, or other conventional agentsfor such use which may be employed in amounts specified in the PDR.

Pharmaceutical Compositions of the Invention

The invention includes a pharmaceutical composition containing apyrrolidine compound of the invention, with or without anothermedicament as described above, in association with a pharmaceuticalcarrier. The pharmaceutical composition can be formulated with one ormore carriers such as conventional solid or liquid vehicles or diluentsand pharmaceutical additives of a type appropriate to the mode ofdesired administration. The pyrrolidine compound of the invention in apharmaceutical composition can be administered to mammalian species,especially humans, an oral, buccal, rectal, pulmonary or similar route,for example, in the form of tablets, capsules, granules or powders. Itcan be administered by a parenteral route in the form of injectablepreparations. It can be administered by a transdermal route either by arelease patch for transdermal delivery or by electro-transport using anappropriate delivery device.

Pharmaceutical compositions containing a pyrrolidine compound of theinvention of the invention may be prepared by conventional techniques,e.g. as described in Remington: The Science and Practice of Pharmacy,19th Ed., 1995. The compositions may appear in conventional forms, forexample capsules, tablets, aerosols, solutions, suspensions or topicalapplications.

A typical pharmaceutical composition includes a pyrrolidine compound ofthe invention formulated with a pharmaceutically acceptable carrierwhich may be an excipient or a diluent, or may be enclosed within acarrier which can be in the form of a capsule, sachet, tablet, paper orother container. In making the composition, conventional techniques forthe preparation of pharmaceutical compositions may be used.

For example, a pyrrolidine compound of the invention will usually bemixed with a carrier, or diluted by a carrier, or enclosed within acarrier which may be in the form of an ampoule, capsule, tablet, sachet,paper, or other container. When the carrier serves as a diluent, it maybe solid, semi-solid, or liquid material that acts as a vehicle,excipient, or medium for the active compound. The pyrrolidine compoundcan be adsorbed on a granular solid container for example in a sachet.Some examples of suitable carriers are water, salt solutions, alcohols,polyethylene glycols, polyhydroxyethoxylated castor oil, peanut oil,olive oil, gelatin, lactose, terra alba, sucrose, dextrin, magnesiumcarbonate, sugar, cyclodextrin, amylose, magnesium stearate, talc,gelatin, agar, pectin, acacia, stearic acid or lower alkyl ethers ofcellulose, silicic acid, fatty acids, fatty acid amines, fatty acidmonoglycerides and diglycerides, pentaerythritol fatty acid esters,polyoxyethylene, hydroxymethylcellulose and polyvinylpyrrolidone.Similarly, the carrier or diluent may include any sustained releasematerial known in the art, such as glyceryl monostearate or glyceryldistearate, alone or mixed with a wax.

A formulation can be mixed with auxiliary agents which do notdeleteriously react with the pyrrolidine compound. Such additives caninclude wetting agents, emulsifying and suspending agents, salt forinfluencing osmotic pressure, buffers and/or coloring substancespreserving agents, sweetening agents or flavoring agents. Apharmaceutical composition can also be sterilized if desired.

The route of administration may be any route, which effectivelytransports the pyrrolidine compound of the invention to the appropriateor desired site of action, such as oral, nasal, pulmonary, buccal,rectal, subdermal, intradermal, transdermal or depot, subcutaneous,intravenous, intraurethral, intramuscular, intranasal, ophthalmicsolution or an ointment, the oral route being preferred.

If a solid carrier is used for oral administration, the preparation maybe tabletted, placed in a hard gelatin capsule in powder or pellet formor it can be in the form of a troche or lozenge. If a liquid carrier isused, the preparation may be in the form of a syrup, emulsion, softgelatin capsule or sterile injectable liquid such as an aqueous ornon-aqueous liquid suspension or solution.

Injectable dosage forms generally include aqueous suspensions or oilsuspensions which may be prepared using a suitable dispersant or wettingagent and a suspending agent. Injectable forms may be in solution phaseor in the form of a suspension, which is prepared with a solvent ordiluent. Acceptable solvents or vehicles include sterilized water,Ringer's solution, or an isotonic aqueous saline solution.Alternatively, sterile oils may be employed as solvents or suspendingagents. Preferably, the oil or fatty acid is non-volatile, includingnatural or synthetic oils, fatty acids, mono-, di- or tri-glycerides.

For injection, a pharmaceutical composition may be a powder suitable forreconstitution with an appropriate solution as described above. Examplesof these include, but are not limited to, freeze dried, rotary dried orspray dried powders, amorphous powders, granules, precipitates, orparticulates. For injection, a pharmaceutical composition may optionallycontain stabilizers, pH modifiers, surfactants, bioavailabilitymodifiers and combinations of these. A pyrrolidine compound of theinvention may be formulated for parenteral administration by injectionsuch as by bolus injection or continuous infusion. A unit dosage formfor injection may be in ampoules or in multi-dose containers.

A pharmaceutical composition of the invention may be designed to providequick, sustained, or delayed release of the active ingredient afteradministration to the patient by employing procedures well known in theart. Thus, a pharmaceutical composition may also be formulated forcontrolled release or for slow release.

A pharmaceutical composition of the invention may include, for example,micelles or liposomes, or some other encapsulated form, or may beadministered in an extended release form or an enteric coated form toprovide a prolonged storage and/or delivery effect. Therefore, thepharmaceutical composition may be compressed into pellets or cylindersand implanted intramuscularly or subcutaneously as depot injections oras implants such as stents. Such implants may employ known inertmaterials such as silicones and biodegradable polymers, e.g.,polylactide-polyglycolide. Examples of other biodegradable polymersinclude poly (orthoesters) and poly (anhydrides).

A pyrrolidine compound of the invention may be formulated as a sustainedrelease implant or implantable material suitable for continuousadministration over a significant period of time. Typical sustainedrelease implants are formed from polymers of pharmaceuticallyacceptable, biodegradable polymers such as polymers and copolymers oflactic acid, lactide, glycolic acid, glycolide, caproic acid andcaprolactone. The dose and amount of pyrrolidine compound of theinvention within the implant will be calculated to deliver the desiredsingle dose blood level of pyrrolidine compound.

For nasal administration, a pharmaceutical composition may contain apyrrolidine compound of the invention dissolved or suspended in a liquidcarrier, in particular an aqueous carrier, for aerosol application. Thecarrier may contain additives such as solubilizing agents, e.g.,propylene glycol, surfactants, absorption enhancers such as lecithin(phosphatidylcholine) or cyclodextrin, or preservatives such asparabenes.

For parenteral application, particularly suitable are injectablesolutions or suspensions, preferably aqueous solutions with apyrrolidine compound of the invention dissolved in polyhydroxylatedcastor oil.

Tablets, dragees, or capsules having talc and/or a carbohydrate carrieror binder or the like are particularly suitable for oral application.Preferable carriers for tablets, dragees, or capsules include lactose,corn starch, and/or potato starch. A syrup or elixir can be used incases where a sweetened vehicle can be employed.

A typical tablet that may be prepared by conventional tablettingtechniques may contain:

Core: Pyrrolidine compound of the invention* 300 mg Colloidal silicondioxide (Aerosil) ® 1.5 mg Cellulose, microcryst. (Avicel) ® 70 mgModified cellulose gum (Ac-Di-Sol) ® 7.5 mg Magnesium stearate AddeCoating: HPMC approx. 9 mg **Mywacett 9-40 T approx. 0.9 mg *Pyrrolidinecompound is formulated as free compound or salt thereof. **Acylatedmonoglyceride used as plasticizer for film coating.

A typical capsule for oral administration contains a pyrrolidinecompound of the invention (250 mg), lactose (75 mg) and magnesiumstearate (15 mg). The mixture is passed through a 60 mesh sieve andpacked into a No. 1 gelatin capsule. A typical injectable preparation isproduced by aseptically placing 250 mg of pyrrolidine compound of theinvention into a vial, aseptically freeze-drying and sealing. For use,the contents of the vial are mixed with 2 mL of physiological saline, toproduce an injectable preparation.

A pyrrolidine compound of the invention may be administered to a mammal,especially a human in need of such treatment, prevention, elimination,alleviation or amelioration of the various malconditions mentionedabove. Such mammals include also animals, both domestic animals, e.g.household pets, farm animals, and non-domestic animals such as wildlife.

A pyrrolidine compound of the invention is effective over a wide dosagerange. For example, in the treatment of adult humans, dosages from about0.5 to about 2000 mg, preferably from about 10 mg to about 1000 mg, perday, more preferably about 50 to 750 mg per day may be used. A typicaldosage is about 50 mg to about 750 mg per day. In choosing a regimen forpatients it may frequently be necessary to begin with a higher dosageand when the condition is under control to reduce the dosage. The exactdosage will depend upon the mode of administration, on the therapydesired, form in which administered, the subject to be treated and thebody weight of the subject to be treated, and the preference andexperience of the physician or veterinarian in charge.

Generally, a pyrrolidine compound of the invention is dispensed in unitdosage form including from about 0.5 to about 2000 mg of activeingredient together with a pharmaceutically acceptable carrier per unitdosage.

Usually, a dosage form suitable for oral, nasal, pulmonary ortransdermal administration includes from about 0.5 mg to about 2000 mg,preferably from about 10 mg to about 1000 mg per day, more preferablyfrom about 50 mg to about 750 mg of a pyrrolidine compound admixed witha pharmaceutically acceptable carrier or diluent.

A pharmaceutical combination of the invention may be formulated as apharmaceutical composition employing all of the embodiments, carriers,route designs and the like described above for formulation of apharmaceutical composition of a pyrrolidine compound alone.

The invention also encompasses prodrugs of a pyrrolidine compound of theinvention which on administration undergo chemical conversion bymetabolic processes before becoming active pharmacological substances.In general, such prodrugs will be functional derivatives of apyrrolidine compound of the invention which are readily convertible invivo into a pyrrolidine compound of the invention. Conventionalprocedures for the selection and preparation of suitable prodrugderivatives are described, for example, in “Design of Prodrugs”, ed. H.Bundgaard, Elsevier, 1985.

The invention also encompasses active metabolites of a pyrrolidinecompound of the invention.

Thus, another aspect of the invention provides a pharmaceuticalcomposition of a pyrrolidine compound of the invention, alone or incombination with another type antidiabetic agent and/or other typetherapeutic agent.

Additional embodiments of the invention are represented by:

-   A pharmaceutical composition including a pyrrolidine compound of the    invention, as described above, together with at least one    pharmaceutically acceptable carrier or diluent;-   Methods of making a pharmaceutical composition of a pyrrolidine    compound of the invention wherein the pharmaceutically acceptable    carrier or diluent is suitable for oral administration;-   Methods of making a pharmaceutical composition of a pyrrolidine    compound of the invention suitable for oral administration further    including the step of formulating the composition into a tablet or    capsule;-   Methods of making a pharmaceutical composition of a pyrrolidine    compound of the invention wherein the pharmaceutically acceptable    carrier or diluent is suitable for parenteral administration;-   Methods of making a pharmaceutical composition of a pyrrolidine    compound of the invention suitable for parenteral administration    further including the step of lyophilizing the composition to form a    lyophilized preparation.

DPP-IV inhibitory activity of the pyrrolidine compound of the inventionmay be determined by use of an in vitro assay system. Inhibitionconstants (Ki or IC50 values) for the DPP-IV inhibitors of the inventionmay be determined by the method described below.

A further detailed description of the invention is given with referenceto the following non-limiting examples.

EXAMPLES Example 1 Synthesis of Pyrrolidine Compounds of the Invention

(2R)-boroPro-(1S,2S,3R,5S)-pinanediol ester, hydrochloride (2): A flamedried round bottom flask equipped with a magnetic stir bar was chargedwith N-Boc-pyrrolidine (20 g, 117 mmol, 1 eq) and dry THF (60 mL) undera nitrogen atmosphere. The clear colorless solution was cooled to −78°C. and a solution of s-BuLi (100 mL of a 1.4 M solution in cyclohexane,140 mmol) was added slowly over a 30 minute period. The light orangecolored solution was stirred at −78° C. for 3 hours followed bytreatment with B(OMe)₃ (39 mL, 350 mmol) after which the cooling bathwas removed and the clear colorless solution slowly warmed to 0° C. Uponreaching 0° C., the reaction was quenched with a small amount of water(˜2 mL), allowed to warm to room temp then extracted into 2 N NaOH (250mL) and backwashed with additional EtOAc (150 mL). The aqueous phase wasacidified to pH 3 by the addition of 2 N HCl and then extracted withEtOAc (3×120 mL). The organic extracts were combined and dried overNa₂SO₄ and concentrated to produce the free boronic acid (22.08 g, 103mmol) as a sticky white solid in 88% yield. Without further purificationthe boronic acid was dissolved in tert-butyl methyl ether (150 mL) andwith constant stirring (+)-pinanediol (17.5 g, 103 mmol) was added atroom temperature. After 18 hr the ether was removed and the(+)-pinanediol boronic ester was purified by column chromatography(silica gel, 1:3 hexanes/EtOAc) to give a clear thick oil (26.84 g, 76.8mmol, 76% yield, R_(f)=0.6 using a 2:1 hexane/ethyl acetate eluant, madevisual via I₂ and/or PMA stain). Removal of the Boc protecting group wasachieved by dissolving the oil in dry ether, cooling to 0° C. in an icebath and with constant stirring dry HCl (g) was bubbled into thesolution for 10 minutes. After 2 hours a white precipitate developed inthe flask and the ether and excess HCl were removed in vacuo to affordthe racemic HCl salt as a white solid. Crystallization and isolation ofthe desired isomer was performed by dissolving the HCl salt in a minimalamount of dichloromethane (250 mL) with gentle heating to facilitate ahomogenous solution followed by continuous stirring for 8 hours to yielda fluffy white precipitate that was collected by vacuum filtration,dried and then dissolved in minimal 2-propanol (˜200 mL) with gentleheating until homogenous. The alcoholic solution was stirred over nightand the resulting white precipitate was collected by vacuum filtrationaffording isomerically pure 2 as a white solid. (7.0 g, 27 mmol, 23%yield). ¹H NMR (400 MHz, D₂O) δ 4.28 (d, J=8.0 Hz, 1H), 3.06 (m, 3H),2.18 (m, 1H), 1.96 (m, 2H), 1.78 (m, 3 H), 1.62 (m, 2H), 1.21 (s, 3H),1.05 (m, 5H), 0.84 (d, J=12 Hz, 2H), 0.71 (s, 2H), 0.62 (s, 3H).

(2R)-1-(2-Chloroacetyl)-boroPro-(1S,2S,3R,5S)-pinanediol ester (3): To asolution of 2 (36.7 g, 129.3 mmol) dissolved in dry CH₂Cl₂ (200 mL)cooled to 0° C. was added chloroacetyl chloride (12.34 mL, 155.2 mmol)under a blanket of N₂. To this was slowly dripped 4-methylmorpholine(42.4 mL, 182 mmol) to give an almost clear light orange solution thatwas warmed to room temp. After 30 minutes the solution was cooled againto 0° C. and 200 mL of a 0.2 N solution of HCl was added and the organiclayers separated, dried and concentrated to give a dark red oil that wasa single spot by TLC (2:1 hex/EtOAc, R_(f)=0.22, made visual via I₂and/or PMA stain) and was used in the next step without furtherpurification. ¹H NMR (400 MHz, CDCl₃) δ0.80 (s, 3 H), 1.25 (m, 1 H),1.26 (s, 3 H), 1.42 (s, 3H), 1.75-1.96 (m, 4 H), 1.98-2.10 (m, 3 H),2.12-2.20 (m, 1 H), 2.29-2.35 (m, 1 H), 3.12-3.16 (m, 1 H), 3.47-3.53(m, 1 H), 3.58-3.63 (m, 1 H), 3.97-4.05 (q, 2 H), 4.30-4.32 (d, 1 H).

(2R)-1-{2-[(3R)-1-tert-Butoxycarbonyl-pyrrolidin-3-ylamino]-acetyl}-pyrrolidine-2-boronicacid (1S,2S,3R,5S)-pinanediol ester (6): Compound 3 (22.7 g, 70 mmol)was dissolved in dry THF (600 mL) followed by addition of excess K₂CO₃and cooled to 0° C. before addition of(3R)-3-amino-pyrrolidine-1-carboxylic acid tert-butyl ester (14.35 g,77.2 mmol). The reaction mixture was warmed to room temperature andstirred for an additional 2.5 days. When TLC indicated all startingmaterial was consumed (10% MeOH in CH₂Cl₂, product was visualized withI₂ stain and appeared as two separate spots (open and closed)R_(f)=0.55). The mixture was filtered through a celite pad, washed with5% MeOH in CH₂Cl₂ (500 mL) and concentrated to yield a sticky, lightlyyellow solid. The solid was dissolved in minimal CH₂Cl₂ followed bycolumn chromatography (silica gel 60, eluted with 5% MeOH in CH₂Cl₂)solution to give 12.1 g of 6. MS m/z (rel intensity) 476 (M+1)⁺ (100),376 (74), 239 (38), 224 (67), 155 (55).

(2R)-1-{2-[(3R)-Pyrrolidin-3-ylamino]-acetyl}-pyrrolidine-2-boronic acid(7): A solution of compound 6 in 4N HCl in dioxane was stirred at roomtemperature for 4 h. The solvent was removed under vacuum and theresulting yellow residue was dissolved in 1N HCl and an equal amount ofhexane. To this bi-phasic solution was added phenyl boronic acid and themixture was stirred vigorously. The hexane layer was periodicallyremoved and replaced with fresh hexane 6 times over a 24-hour period andprogress monitored via LCMS. The aqueous layer was separated and appliedto a Dowex 50-X2-100 ion exchange column (H+ form) and eluted with wateruntil the eluant was neutral. Then the eluant was changed to aqueousammonium hydroxide (2% vbw) followed by lyophilization of theappropriate fractions to yield 7 as a white crystalline solid (Table 1).TFA salt ¹H-NMR (500 MHz, CD₃OD) δ 4.13 (m, 1H), 4.08 (bs, 2H), 3.76(dd, J=13.0, 8.0 Hz, 1H), 3.55 (m, 3H), 3.41 (m, 2H), 3.27 (m, 1H), 2.53(m, 1H), 2.26 (m, 1H), 2.10 (m, 2H), 1.99 (m, 1H), 1.75 (m, 1H). MS m/z(rel intensity) 224 (M−17) (100), 206 (25), 180 (29), 155 (70).

(2R)-1-{2-[(3S)-1-tert-Butoxycarbonyl-pyrrolidin-3-ylamino]-acetyl}-pyrrolidine-2-boronicacid (1S,2S,3R,5S)-pinanediol ester (8): The protocol described abovefor the synthesis of 6 was followed employing(3S)-3-amino-pyrrolidine-1-carboxylic acid tert-butyl ester in place of(3R)-3-amino-pyrrolidine-1-carboxylic acid tert-butyl ester. Compound 8was obtained as an oil. MS m/z (rel intensity) 476 (M+1)⁺ (100), 376(74), 239 (38), 224 (67), 155 (55).

(2R)-1-{2-[(3S)-Pyrrolidin-3-ylamino]-acetyl}-pyrrolidine-2-boronic acid(9): The protocol described above for the synthesis of 7 was followed toproduce 9 (table 1). 8•TFA salt ¹H-NMR (500 MHz, CD₃OD) δ 4.12 (m, 3H),3.76 (m, 1H), 3.54 (m, 3H), 3.41 (m, 2H), 3.26 (m, 1H), 2.55 (m, 1H),2.28 (m, 1H), 2.05 (m, 3H), 1.74 (m, 1H). MS m/z (rel intensity) 241 (M)(27), 224 (100), 209 (73), 155 (47).

TABLE 1 Compound No. Name Structure LC-MS 7 (2R)-1-{2-[(3R)-Pyrrolidin-3- ylamino]-acetyl}- pyrrolidine-2- boronic acid

242 (M + 1)(100), 224 (9) 9 (2R)-1-{2-[(3S)- Pyrrolidin-3-ylamino]-acetyl}- pyrrolidine-2- boronic acid

242 (M + 1)(100), 224 (19)

Example 2 DPP-IV Inhibitory Assays

The purification of porcine DPP-IV and the enzyme assay under steadystate conditions are described in (1) Rahfeld, J. Schutkowski, M.,Faust, J., Neubert., Barth, A., and Heins, J. (1991) Biol. Chem.Hoppe-Seyler, 372, 313-318; and (2) Nagatsu, T., Hino, M., Fuyamada, H.,Hayakawa, T., Sakakibara, S., Nakagawa, Y., and Takemoto, T. (1976)Anal. Biochem., 74, 466-476, respectively. Human DPP-IV is alsocommercially available from, e.g., Research Diagnostics.

The activity of human DPP-IV was measured in vitro by its ability tocleave the synthetic substrate Gly-Pro-AMC. Cleavage of Gly-Pro-AMC byDPP-IV liberates the product AMC (7-amino-4-methyl coumarin), whose rateof appearance is directly proportional to the enzyme activity.Inhibition of the enzyme activity by specific enzyme inhibitors slowsdown the generation of AMC. Stronger interaction between an inhibitorand the enzyme results in a slower rate of generation of AMC. Thus, thedegree of inhibition of the rate of accumulation of AMC is a directmeasure of the strength of enzyme inhibition. The accumulation of AMC ismeasured fluorometrically. The IC₅₀ (concentration of test compound atwhich 50% of the enzyme activity is inhibited) for each compound isdetermined by incubating fixed amounts of enzyme with several differentconcentrations of inhibitor. The inhibition constant, Ki, can bedetermined for each compound by incubating fixed amounts of enzyme withseveral different concentrations of inhibitor and substrate.

The compounds of the invention were tested for their ability to inhibitthe activity of purified DPP-IV using the following methods. DPP-IVenzyme activity was determined by a fluorometric assay with thesubstrate Gly-Pro-AMC which is cleaved by DPP-IV to release thefluorescent AMC leaving group. Free AMC (7-amino-4-methyl coumarin) wasmeasured using an excitation wavelength of 380 nm and an emissionwavelength of 460 nm with a Victor-II fluorescent reader. Stocksolutions of DPP-IV (1 ng/μl, pH 8.0) and Gly-Pro-AMC substrate (400 μM)in 25 mM Tris buffer (pH 8.0) were prepared separately. Test compoundswere dissolved in DMSO or in 50 mM glycine buffer (pH 3.0). The assaywas performed by diluting the DPP-IV stock (10 μl) into 25 mM Trisbuffer (77.5 μl) followed by addition of test compound (2.5 μl) at 26°C. After 10-minutes substrate was added (10 μl) and allowed to react for20-minutes at 26° C. before free AMC was measured. IC₅₀ values weredetermined in triplicate, using a minimum of six different inhibitorconcentrations. IC₅₀ values were calculated using Nonlinear RegressionAnalysis (GraphPad, Prism, San Diego, Calif.).

Compounds 7 and 9 were tested in vitro as inhibitors of DPP-IV asdescribed herein and each displayed an IC₅₀ of 1 μM or less.

Example 3 DPP-VII, DPP-VIII, DPP-IX, and FAP Inhibitory Assays

Recombinant DPP-VII, DPP-VIII, DPP-IX, and FAP enzymes were prepared asfollows. The full length cDNAs for human DPP-VII, DPP-VIII, DPP-IX, andFAP were obtained from Open Biosystems. The cDNAs were cloned into thepFastBac vector with the addition of an N-terminal FLAG tag on DPP-VII,C-terminal 6×His tags on DPP-VIII and DPP-IX, and an N-terminal 6×Histag on FAP (Sun 2002, Qi 2003, and Chen 2004). Baculovirus was preparedusing the Bac-to-Bac Baculovirus Expression System (Invitrogen). ThecDNAs in the final baculovirus constructs were sequence verified.

To express the recombinant DPPs from the baculovirus constructs, S9insect cells (cells and Sf-900 SFM media purchased from Invitrogen) weregrown to mid log phase at 27° C. with shaking at 125 rpm and thenadjusted to 2×10E6/ml just prior to baculovirus infection. Infectionwith DPP-VII, DPP-VIII, DPP-IX, and FAP baculoviral constructs were allperformed at an MOI of 4. The infected cells were grown for 48 hours andthe cell pellets harvested and frozen until purification. DPP-VII waspurified using anti-FLAG immunoaffinity gel according to themanufacturer's instructions. DPP-VIII, DPP-IX, and FAP were purifiedusing a B-PER 6×His Fusion Protein Column Purification Kit from Pierce.

The activity of DPP-VII, DPP-VIII, DPP-IX and FAP was measured in vitroby its ability to cleave the synthetic substrates Lys-Pro AMC (DPP-VII)and Ala-Pro AMC (DPP-VIII, DPP-IX and FAP) (substrates purchased fromEnzyme Systems). Recombinant DPPs were diluted in reaction buffer togive fluorescence values of 5000-20000 counts in the “enzyme only wells”20 min after addition of substrate at 27° C. Reaction buffers were 25 mM(2-(4-Morpholino)-Ethane Sulfonic Acid), pH 5.5 for DPP-VII, 25 mM Tris,pH 8, 1% Triton X-100, 100 mM NaCl for DPP-VIII, and 25 mM Tris pH 8 forDPP-IX and FAP. Test wells in a 96-well microtiter plate contained 88 μLof diluted DPP and 2.5 ul of titrated compound in 50 mM glycine, pH 2.6.“Enzyme only” wells contained 88 uL of diluted DPP and 2.5 ul of glycinebuffer. “No enzyme” wells contained 88 uL of reaction buffer without DPPand 2.5 ul of glycine buffer. All assays were done in triplicate. Theplate was incubated at 27° C. for 10 min and then cooled on ice for 10min. Ten microliters of substrate diluted in reaction buffer (40 uMfinal concentration) without Triton or NaCl were then added to all wellsfollowed by incubation at 27° C. for 20 min. Fluorescence in each wellwas measured at settings of 380/460 nm. IC₅₀ calculations were performedby non-linear regression analysis using Prism software (GraphPad).

Example 4 DPP Selectivity of Boronic Acids

Using the methods described above, the DPP-IV, VII, VIII, IX and FAPinhibitory activities of compounds 7 and 9 were compared to the closelyrelated analogues shown in Table 2.

TABLE 2 Other Boronic Acid Inhibitors Compound Number Structure 5

14

15

16

17

18

19

20

The selectivity ratio value was obtained by dividing the IC₅₀ value forDPP-VII, DPP-VIII, DPP-IX or FAP by the IC₅₀ value for DPP-IV andassigned a selectivity ratio defined as A≦1; 1≦B≦10; 10<C≦50; D>50.Results are summarized in Table 3.

TABLE 3 DPP Selectivity Ratios Compound Selectivity Ratios* No. DPP-VIIDPP-VIII DPP-IX FAP 5 A B A B 7 C D C D 9 C C A C 14 A B A D 15 A B A C16 B C A C 17 B B A C 18 B C A C 19 A B B B 20 A A A B *Selectivityratios: A ≦ 1; 1< B ≦ 10; 10< C ≦ 50; D > 50

Compounds 7 and 9, incorporating pyrrolidine, show greatly improvedselectivity in contrast to their closely related analogues. For example,compounds 7 and 9 show excellent selectivity for DPP-IV relative toDPP-VIII. Replacement of the 3-aminopyrrolidine with cyclopentyl 5,4-piperidinyl 14, 3R-piperidinyl 15, or 3S-piperidinyl 16 (Table 2)maintains inhibition of DPP-IV. However, compounds 5, 14, 15, and 16 aresignificantly less selective towards the other dipeptidyl peptidaseswith one or more exhibiting selectivity ratios ≦1; and two or moreexhibiting selectivity ratios ≦10. Thus, examples of the claimedcompounds (compound 7 and 9) demonstrate unexpected selectivity towardsthe other dipeptidyl peptidases while maintaining potency againstDPP-IV, while the close structural analogues of the selective compoundsof the invention exhibit significant activity against one or more of theother dipeptidyl peptidases.

Also, addition of further moieties at positions on the pyrrolidine ringproduces compounds with reduced selectivity towards the other dipeptidylpeptidases. Alkylation of the pyrrolidine nitrogen yielding compounds 17and 18, acylation of the pyrrolidine nitrogen yielding compound 19 orincorporation of a carboxamide yielding compound 20 all lead to asubstantial loss of selectivity. See Table 2 for the structures of thesecompounds.

Example 5 In vivo Toxicity of Boronic Acids

To determine toxicity of boronic acids in rats, the Zucker DiabeticFatty (ZDF) rat model was used. After overnight fasting, adult rats wereorally administered either vehicle or test compound at the indicateddoses once a day and twice a day. Animals were monitored for clinicalsigns at 1, 2, 4 and 8 hour time intervals following the treatmentregimen and blood samples were collected. Oral doses of compound 5 at 10mg/kg and PT100 (valboropro, Bachovchin et al., PNAS, 88:1556, 1991;Bachovchin et al. J. Med. Chem, 39:2087, 1996) at 1 mg/kg caused acutetoxicity (death) 4-24 hrs post-dose in all Zucker Obese Fatty ratstested. By contrast, inventive compound 7 was well tolerated at doses upto 80 mg/kg (≧90% DPP-IV inhibition for 24 hrs at all doses).

To assess toxicity of boronic acids in dogs, IV dosing studies wereperformed. Compound 7, of the invention, was compared with the closelyrelated analog, compound 5. Compound 5 is a potent inhibitor of DPP-IVand all the other DPPs, whereas compound 7 is a potent and selectiveinhibitor of DPP-IV. When compound 5 was administered to dogs at 0.2mg/kg or higher, increasing toxicity was observed with severe emesis anddiarrhea observed at 2 mg/kg while a dose of 0.1 mg/kg did not elicitany toxicities. A comparison of the plasma concentrations of 5 at 0.1and 0.2 mg/kg demonstrated that the time above the DPP-VIII IC₉₀ was 2and 4 hours respectively and greater than 14 hours for DPP-IX, the DPPwith the lowest selectivity ratio (Table 3). This observation coupledwith the 2-4 hour onset of toxicity demonstrates that activity againstDPP-VIII mediates the observed toxicity. Compound 7, a selective DPP-IVinhibitor, when administered at a dose of (6.0 mg/kg), a dose thatresulted in plasma levels at the IC₉₀ for DPP-VIII for less than 2hours, showed no toxicity confirming the improved safety of compounds ofthe invention over non-selective DPP inhibitors.

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements will be apparent to thoseskilled in the art without departing from the spirit and scope of theclaims.

All patents and publications are herein incorporated by reference to thesame extent as if each individual publication was specifically andindividually indicated to be incorporated by reference.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

1. A pharmaceutical composition comprising a pharmaceutically acceptablecarrier and a therapeutically effective amount of a compound of formula(I)

or pharmaceutically acceptable salt thereof; wherein R² and R³independently or together are —OH, —O⁻M⁺wherein M⁺is a cation, ahydroxyl bearing a boronic acid protecting group, or a group capable ofbeing hydrolyzed to a hydroxyl group in an aqueous solution atphysiological pH or in biological fluids, and wherein the compound offormula (I) is a selective inhibitor of DPP-IV.
 2. The pharmaceuticalcomposition of claim 1, wherein R² and R³ are both —OH and wherein thecompound of formula (I) is:

or a pharmaceutically acceptable salt thereof.
 3. The pharmaceuticalcomposition of claim 1 or 2, wherein the composition is an oral dosageform.
 4. The pharmaceutical composition of claim 1 or 2 wherein thetherapeutically effective amount is about 50 mg to about 750 mg.